PREFORMULATION PARAMETERS CHARACTERIZATION TO DESIGN, DEVELOPMENT AND FORMULATION
OF LOXOPROFEN LOADED MICROSPHERES
P.Venkatesan*, V. Sree Janardhanan, R.Manavalan, K.Valliappan
Department of Pharmacy, Faculty of Engineering and Technology, Annamalai University, Annamalainagar, TN 608 002, India
Abstract
The purpose of the present study was to systematically investigate some of the important physicochemical properties of loxoprofen loaded microspheres. Loxoprofen, 2-[4-(2-oxocyclopentylmethyl) phenyl]-propionate with two chiral centers, is marketed as an equal parts mixture of four stereoisomers. Loxoprofen sodium is an important non-steroidal anti-inflammatory drug (NSAID) of the 2-arylpropionic acid group used for the treatment of rheumatoid arthritis and osteoarthritis. Almost all drugs are marketed as tablets, capsules or both.
Prior to the development of these major dosage forms, it is essential that pertain fundamental physical and chemical properties of the drug molecule and other divided properties of the drug powder are determined. This information decides many of the subsequent events and approaches in formation development.A sustained release microsphere of Loxoprofen was prepared by solvent evaporation method using ethyl cellulose as coating material. So before selection of excipients, the Preformulation study of drug loxoprofen is completed for successful formulation of sustained release microspheres. Preformulation studies included solubility, pKa, dissolution, melting point, assay development, stability in Solution, stability in solid state; microscopy, bulk density, flow properties, excipient compatibility, entrapment efficiency and release profile of microspheres were investigated.
Key words: Microspheres, entrapment efficiency, Loxoprofen, Sustained release, preformulation
INTRODUCTION
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain and inflammation. NSAIDs produce their therapeutic effect by inhibiting the cyclooxygenase (COX) enzymes, which are involved in the biosynthesis of prostaglandins (PGs) [1, 2]. Loxoprofen, 2-[4-(2- oxocyclopentylmethyl) phenyl]-propionate with two chiral centres, is marketed as an equal parts mixture of four stereoisomers. Loxoprofen sodium is an important non-steroidal anti-inflammatory drug (NSAID) of the 2- arylpropionic acid group used for the treatment of rheumatoid arthritis and osteoarthritis. Loxoprofen is a prodrug which produces effects after being absorbed from the gastrointestinal tract followed by conversion to an active metabolite. The mechanism of action of loxoprofen is due to inhibition of prostaglandin biosynthesis by its action on cyclooxygenase. After oral administration, loxoprofen sodium is absorbed as the free acid from the gastrointestinal tract rather than the sodium salt, which causes just weak irritation of the gastric mucosa, and is then converted to an active metabolite by reduction of the ketone carbonyl to the trans-OH form. The active isomer has the 2S, 1R', 2'S configuration, which potently inhibits prostaglandin biosynthesis [3–5]. Loxoprofen has an activity to treat inflammatory rheumatoid diseases and relieve acute pain. It is effective against period pains, pain after surgery and fever. Loxoprofen available in pharmaceutical formulations as Tablets and Transdermal patches [6, 7].
Preformulation commences when a newly synthesized drug shows sufficient pharmacologic promise in animal models to warrants evaluation in man. These studies should focus on those physicochemical properties of the new compound that could affect drug performance and development of an efficacious dosage form. A thorough understanding of these properties may ultimately provide a rational for formulation design, or support the need for molecular modification[8].
The aim of this study was to determine some of the physicochemical properties such as solubility, pKa, dissolution, melting point, assay development, stability in Solution, stability in solid state, microscopy, bulk density, flow properties, excipient compatibility, entrapment efficiency and release profile of microspheres Materials and Methods
Loxoprofen (99.79%) and Ethylcellulose (viscosity grade, 100 cP) were donated by M/S Micro labs limited, Hosur. India. Dichloromethane, tween 80, acetonitrile (ACN) of HPLC grade and dipotassium hydrogen phosphate and phosphoric acid were of analytical-reagent grade supplied by M/S SD Fine chemicals, Mumbai, India. The HPLC grade water was prepared by using Milli-Q Academic, Millipore, and Bangalore, India. A Remi mechanical stirrer (Remi Motors, India) was used for the preparation of the microspheres. Shimadzu (Tokyo, Japan) model which consisted of a LC10AD and LC10 ADvp solvent delivery module, SPD 20 PDA detector, a Rheodyne injector (model 7125, USA) valve fitted with a 20 µl loop, and PDA detector (SPD-20).
The system was controlled through a system controller (SCL-10A) and a personal computer using a Shimadzu chromatographic software (LC Solution, Release 1.11SP1) installed on it was used for the assay of Loxoprofen.
All chemicals used in the study were of analytical grade and used without further purification.
Experimental Studies Determination of solubility
The loxoprofen sodium was evaluated for solubility in water, acetone, methanol, diethyl ether chloroform and ethanol in accordance with the British pharmacopoeia specifications [9, 10].
pH Determination
This was done by shaking a 1%w/v dispersion of the sample in water for 5min and the pH determination using a digital pH meter (model 335, Systronics, India) [11]. The data presented here is for triplicate determinations.
True density
True density of loxoprofen was determined by liquid displacement method. It is calculated from the volume of intrusion fluid (toluene) displaced in the pycnometer by a given mass of powder [8].
D =
Where, D is true density, Vp is the total volume of the pycnometer and Vi is the volume of intrusion fluid in the pycnometer containing the mass of powder (M). All the estimations were done in triplicate and average are reported in table 1.
Determination of bulk density, bulkiness and compressibility index
The bulk density of loxoprofen was determined by the three tap method [12]. 10g of loxoprofen powder was carefully introduced into a 100 ml graduated cylinder. The cylinder was dropped onto a hard wood surface 3 times from a height of 1inch at an interval of 2 seconds. The bulk density was obtained by dividing the weight of the sample by volume of the sample contained in the cylinder. Reciprocal of bulk density or the specific bulk volume gave the bulkiness. The percent compressibility index (I) [13] of the loxoprofen was calculated using following formula and the results are given in Table1.
I= 1 − x100 Angle of repose
The static angle of repose, a, was measured according to the fixed funnel and free standing cone method. [14] a funnel was clamped with its tip 2cm above a graph paper placed on a flat horizontal surface. The powders were carefully poured through the funnel until the apex of the cone thus formed just reached the tip of the funnel. The mean diameters of the base of the powder cones were determined and the tangent of the angle of repose calculated using the equation:
Tan a = 2h/D The data presented here is for triplicate determinations.
Determination of Partition Coefficient
10 mg drug was added in 50 ml of n-Octanol (pre saturated with water) and it was shaked and then 50 ml of distilled water (pre saturated with n- Octanol ) was added and was shaked the mixture by mechanical shaker for 24 hours. After 24 hour both phases are separated. Absorbance was taken of both the phases and calculated the concentration in each phases [15].
Partition Coefficient = Percentage of moisture loss
The Loxoprofen loaded microspheres were evaluated for percentage of moisture loss which sharing an idea about its hydrophilic nature. The microspheres weighed initially and kept in desiccator containing calcium chloride at 37 °C for 24 hours. When no further change in weight of sample was observed, the final weight was noted down [16, 17].
% of moisture loss = × 100
Table 1: physicochemical properties of loxoprofen
Parameters
Results
Description Loxoprofen Sodium Hydrate occurs as white to yellowish white crystals or crystalline powder.
Solubility It is very soluble in water and methanol, freely soluble in ethanol(95), practically in soluble in diethyl ether, acetone and chloroform
pH 7.1 ± 0.27
True density (gm/cc) 1.62 ± 0.42 Bulk density (gm/cc) 0.64 ± 0.57 Compressibility Index (%) 13.17 ± 0.35 Angle of repose ( o ) 33.16 ± 1.15 Moisture content (%) 8.52 ± 0.45 Partition Coefficient 1.87 ± 0.53
Determination of Particle Size distribution
5ml of 1%w/v dispersion of loxoprofen in glycerine was prepared. A smear of the dispersion was made and examined under microscope. The size of 500 particles was measured using a calibrated eyepiece micrometer [18, 19]. The size distribution of the loxoprofen particles was estimated. The results are given in Table 2 and shown in Fig.1
Table 2 Particle size distribution of loxoprofen powder
Size range(µm) Number of particles
0-30 22 30-60 112 60-90 215 90120 124
>120 27
Fig. 1: Particle size distribution of Loxoprofen
STABILITY OF LOXOPROFEN IN SOLVENTS:
Stability of Loxoprofen in distilled water
Stability of Loxoprofen in distilled water was determined by keeping the known concentration (40 µg/
ml), in kinetic mode of UV –Visible spectrophotometer (UV – 160IPC, Shimadzu, Japan) for 30 minute [20] . The data obtained is shown in Table 3.
Table 3: Stability of Loxoprofen in Distilled Water in Kinetic Mode of UV- Visible Spectrophotometer at ƛ max 220.0 nm (Absorbance of 20 µg/ ml drug solution is 0.6112)
Time (Second) Absorbance dA
0 0.6112 0.000
200 0.6112 0.000 400 0.6110 0.002 600 0.6111 0.001 800 0.6112 0.000 1000 0.6113 -0.001 1200 0.6111 0.001 1400 0.6109 0.003 1600 0.6110 0.002 1800 0.6112 0.000 0
50 100 150 200 250
0-30 30-60 60-90 90-120 >120
Particle size distribution of loxoprofen powder
Size range (µm) Num
ber of parti
cles
Stability of Loxoprofen in 0.1N HCl
This was determined by keeping the known concentration (20 µg/ ml) solution, in kinetic mode of UV – Visible double beam spectrophotometer (UV – 160 IPC, Shimadzu, Japan) for 30 minute. The data obtained is shown in Table 4.
Table 4: Stability of Loxoprofen in 0.1N HCl in Kinetic Mode of UV – Visible Spectrophotometer at ƛ max 220.0 nm (Absorbance of 20 µg/ ml of drug solution is 0.409)
Time (Second) Absorbance dA
0 0.409 0.000
200 0.409 0.000 400 0.407 0.002 600 0.406 0.003 800 0.408 0.001 1000 0.409 0.000 1200 0.409 0.000 1400 0.408 0.001 1600 0.407 0.002 1800 0.408 0.001
Preparation of Calibration Curve of Loxoprofen in Distilled Water:
A stock solution (100 mg / ml) of loxoprofen prepared and from this different –different concentration solutions were prepared and then the absorbance of dilutions was measured on a UV- Visible spectrometer (UV – 160 IPC, Shimadzu, Japan) at 220.0 nm [21]. The absorbance data are given in the Table 5 and the calibration curve is plotted is shown in Fig. 2
Table 5: Absorbance Data for Calibration Curve of Loxoprofen in Distilled Water at 220.0 nm
Concentration (µg/ml)
Absorbance
Set 1 Set 2 Set 3 Average ± SD
0 0.000 0.000 0.000 0.000
20 0.611 0.614 0.612 0.611 ± 0.003
40 1.197 1.197 1.198 1.197 ± 0.001
60 1.756 1.758 1.756 1.756 ± 0.002
80 2.312 2.311 2.315 2.312 ± 0.003
100 2.834 2.834 2.835 2.834 ± 0.001
Fig. 2: Calibration Curve of Loxoprofen in Distilled Water at 220. nm
Determination of interference of additives in the estimation of Loxoprofen
The highest concentration of the additives that would be present in 900 ml of dilution media was estimated on the basis of amount present in per tablet of different batches and the same quantities of different additives were taken in 10 ml volumetric flasks containing 10 ml of 20 µg / ml concentration of Loxoprofen solution. The flasks were kept for 24 hours with occasional shaking and filtration was done. The absorbance of filtrate were measured at 220.0 nm on UV – Visible spectrophotometer (UV – 160IPC, Shimadzu, Japan) and compared with the absorbance of control drug sample of 20 µg / ml concentration without additives [20].
Observations are shown in Table 6:
Table 6: Interference of Additives in the Estimation of Loxoprofen in Distilled Water at 220.0 nm (Absorbance of plain drug solution (20µg / ml): 0.612 A)
S.No. Additives Absorbance Interference
01 Ethyl cellulose 0.612 Nil
02 cellulose acetate phthalate 0.610 Nil
03 Eudragit RS 0.615 Nil
04 Eudragit RL 0.610 Nil
05 Magnesium Stearate 0.610 Nil
06 PVA (%w/v) 0.609 Nil
07 Hydroxy propyl Methyl cellulose
0.610 Nil
Dissolution Test
In-vitro dissolution studies were carried out using a dissolution apparatus USP (Paddle type) at a paddle speed of 50 rpm. The dissolution medium was 900 ml of phosphate buffer, pH 7.4, which was
y = 0.0283x + 0.0352 R² = 0.9993
0 0.5 1 1.5 2 2.5 3 3.5
0 20 40 60 80 100 120
Ab sor ba nc e
Concentration mcg/ml
maintained at 37 ± 0.5 °C. 5 ml of dissolution samples were withdrawn and replaced with equal volume fresh phosphate buffer, pH 7.4 at regular intervals. Collected dissolution samples were used for determination of released loxoprofen concentrations by using a UV-VIS spectrophotometer (UV – 160IPC, Shimadzu, Japan) at 220 nm wavelength against a blank [22].
The dissolution rate of loxoprofen of tablets was over 85% after 30 min (Fig. 3), which conformed to the loxoprofen sodium standards written in the Japanese Pharmaceutical Codex (JPC), part 3.
Fig. 3 : Dissolution rate of loxoprofen
Preparation of Microspheres
This is the method widely used in the microencapsulation process. Calculated quantity of polymer was dissolved in Dichloromethane to form a homogenous polymer solution. Then calculated quantity of drug was added to the polymer solution and mixed thoroughly. The resulting mixture was then added in a thin stream of 300ml of aqueous solution containing 1% (v/v) tween80, while stirring at 1000rpm to emulsify the added dispersion as fine droplets. The solvent, dichloromethane was then removed by evaporation during continuous stirring at room temperature for 3 hours to produce spherical microspheres. Here dichloromethane was used as polymer solvent, aqueous solution as the microencapsulating vehicle, tween80 as the dispersing agent. During 3hours stirring period, dichloromethane was completely removed by evaporation [23-29]. The microspheres were collected by vacuum filtration and washed repeatedly and dried in room temperature over a night to get free flowing microspheres.
Percentage yield
The dried microspheres were weighed and percentage yield of the prepared microspheres was calculated by using the following formula [30, 31]. The results are given in Table 7
Percentage yield = × 100 Scanning electron microscopy (SEM)
The formulated microspheres were taken for the shape and surface characteristic studies. The microspheres were scanned using scanning Electron microscopy, the microspheres were mounted directly on to the scanning Electron Microscopy samples stub using double sided sticking tape, coated with gold in quick auto coater, with a thickness of 200nm under reduced pressure of 0.001 torr. The shape and surface characteristic of the microspheres were observed in Electron micro analyser and photographs were taken using JSM 6400 camera Fig.4
0 20 40 60 80 100 120
0 5 10 15 20 25 30 35 40 45
Time in Minutes Con
cent rati
on
Fig. 4: SEM image of formulation (F3)
Particle Size Analysis of Microspheres
Average particle diameter and size distribution of microspheres were determined by laser diffractometry using a Mastersizer 2000 (Malvern Instruments, Malvern, UK). Approximately 10 mg of microspheres were dispersed in 2 to 3 ml distilled water containing 0.1% Nonidet P40 for several minutes using an ultrasonic bath. Then, an aliquot of the microsphere suspension was added into the small volume recirculation unit [32], which was subsequently circulated 3500 times per minute. Each sample was measured in triplicate for the analysis. Particle size was expressed as the weighted mean of the volume distribution. The results are given in Table 7 and shown in Fig.5
Fig. 5: Particle size of Loxoprofen loaded sustained release microspheres
Encapsulation efficiency (EE)
Drug loaded microcapsules (100 mg) were powdered and suspended in water and then sonicated for about 20 minutes. It was shaken for another 20 minutes for the complete extraction of drug from the microcapsules. The mixture was filtered through a 0.45 μm membrane filter (MILLIPORE). Drug content was
Loxoprofen microspheres with ethyl cellulose formulation (F3)
determined by UV- visible spectrophotometer (UV – 160IPC, Shimadzu, Japan) at 220 nm. The percent entrapment was calculated using the following formula [33]. The results are given in Table 7,
Encapsulation efficiency = × 100 Invitro drug release
The USP XXIV dissolution rate testing apparatus was employed to study the release of ibuprofen using phosphate buffer PH 7.4 as a dissolution medium.100mg equivalent of loxoprofen containing ethyl cellulose microspheres was filled in hard gelatine capsule and dissolution test was being carried out at 50 rpm maintained at 37oC±0.5oC. 5ml of sample were withdrawn at specific time interval for 8hours. The sample volume was replaced by an equal volume of fresh medium. The concentration was determined by UV- visible spectrophotometer (UV – 160IPC, Shimadzu, Japan) at 220 nm. The percentage of drug release at various time intervals was calculated and plotted against time [34-39]. The results are given in Table 7 and shown in Fig.4
Table 7: Loxoprofen microspheres evaluation
Formulation Particle Size % yield Entrapment efficiency % Drug release
F1 196.50 87.34 84.57 86.07
F2 201.10 86.50 85.23 85.81
F3 203.42 85.47 83.52 86.46
Figure 4: Drug Release Profile of formulated loxoprofen microspheres
Stability studies
The microspheres was taken in a crucible and placed at 450C and 75%RH for 45 days, the microspheres were analyzed for their drug content and dissolution studies.
0 10 20 30 40 50 60 70 80 90 100
0 1 2 3 4 5 6 7 8 9
Time in Hours Con
cent rati
on
Table: 8 Stability data for formulation (For-1, Ratio1:3) at 45 ±1ºC
S.NO Time in days Physical changes Percentage drug content (mg)
01 0 --- 86.07
02 15 No change 86.04
03 30 No change 86.07
04 45 No change 86.08
RESULTS AND DISCUSSION
Particle size distribution of drug has influence on many bulk properties of pharmaceutical interest such as flow properties, packing, packing densities, compressibility segregation characteristics etc. Hence, it must be the aim of pharmaceutical technologist to study the particle size distribution. One of the most common methods for particle size determination is optical microscopy, because it gives direct measurement of the individual particle. The particle size distribution of loxoprofen powder was given in Table 2 and shown in Fig.1 and loxoprofen microspheres was given in Table 7 and shown in Fig.5.
The results of solubility, true density, bulk density, compressibility index, angle of repose, moisture content, pH, Partition Coefficient are given in Table 1.
The microspheres had good spherical geometry as evidenced by the SEM photographs. The surface of the microspheres was quite smooth (figure4).
The results of percentage yield, particle size, entrapment efficiency and invitro drug release of formulated sustained release microspheres are given in table 7.
CONCLUSION
The preformulation phase is a critical phase in establishing the properties of CDs that will allow suitable risk assessment for development. Typically it begins during the lead optimization phase, continues through prenomination, and on into the early phases of development. Decisions made on the information generated during this phase can have a profound effect on the subsequent development of those compounds.
Therefore, it is imperative that preformulation should be performed as carefully as possible to enable rational decisions to be made. The quantity and quality of the drugs can affect the data generated as well as the equipment available and the expertise of the personnel conducting the investigations.in this study we successfully completed the physicochemical characterization of Loxoprofen Sodium properties like morphology, size, solubility, pH, Partition coefficient, Surface area flow property, drug content and release study. This knowledge can be useful in developing modified release formulations mainly sustained release formulation of loxoprofen loaded microspheres.
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